Mitochondrial permeability transition (MPT) is an inner membrane permeabilization event, which can result in irreversible de-energization and swelling of mitochondria, leading to release of pro-death factors. Mitochondrial Ca2+ overload is the best-characterized trigger of MPT and has been implicated in the pathogenesis of diverse paradigms of neuronal death, such as ischemia-reperfusion injury, where a large influx of cytosolic Ca2+ triggers mitochondrial Ca2+ overload. While uncontrolled MPT can result in mitochondrial disruption, under certain conditions, MPT could provide mitochondria with a Ca2+ release outlet, allowing Ca2+ recycling and protecting mitochondria from Ca2+ overload. Estrogen receptors (ER) have been implicated in various paradigms of neuronal injury, and MPT modulation could be one of the mechanisms whereby they exert their role. Our studies revealed an unprecedented role of the ER? in modulating MPT. In mouse brain mitochondria, estrogen decreases mitochondrial Ca2+ capacity in an ER? and cyclophilin-D (CyPD, an MPT activator) dependent manner. Mitochondria from ER? knock out (ER?KO) mice have reduced sensitivity to cyclosporine A, a potent CyPD inhibitor and CyPD genetic ablation in ER?KO does not further increase Ca2+ capacity. These results point to ER? as a novel regulator of Ca2+-dependent MPT that functionally interacts with CyPD. In this application, we will test the hypothesis that ER? localized in mitochondria (mER?) regulates MPT, independently of transcriptional effects. The goals are to investigate the mechanisms of MPT modulation by mER? and to test the effects of MPT modulation by mER? in models of neuronal injury that involve mitochondrial Ca2+ toxicity, such as oxygen glucose deprivation (OGD) and glutamatergic toxicity. To this end we propose 1) to study the mechanisms of regulation of Ca2+-mediated MPT by ER?. This regulation will be investigated using a multipronged approach, involving biochemical and molecular studies. 2) To assess the role of ER? MPT regulation in neuronal Ca2+-mediated injury. Evidence suggests that Ca2+ dependent MPT and its regulator CyPD are involved in ischemic neuronal injury. We will use neuronal OGD and exposure to glutamatergic agents, both well-known paradigms of neuronal toxicity involving mitochondrial Ca2+ overload, to test the effects of genetic and pharmacological modulation of ER?. The impact of the project will be two-fold: first, it will elucidate novel mechanisms of MPT regulation; second, it will assess if MPT modulation by mER? could be protective in neuronal injury.